Hydrogel dressing integrating FAK inhibition and ROS scavenging for mechano-chemical treatment of atopic dermatitis

Atopic dermatitis (AD) is a chronic skin disease caused by skin immune dyshomeostasis and accompanied by severe pruritus. Although oxidative stress and mechanical scratching can aggravate AD inflammation, treatment targeting scratching is often overlooked, and the efficiency of mechano-chemically synergistic therapy remains unclear. Here, we find that enhanced phosphorylation of focal adhesion kinase (FAK) is associated with scratch-exacerbated AD. We then develop a multifunctional hydrogel dressing that integrates oxidative stress modulation with FAK inhibition to synergistically treat AD. We show that the adhesive, self-healing and antimicrobial hydrogel is suitable for the unique scratching and bacterial environment of AD skin. We demonstrate that it can scavenge intracellular reactive oxygen species and reduce mechanically induced intercellular junction deficiency and inflammation. Furthermore, in mouse AD models with controlled scratching, we find that the hydrogel alleviates AD symptoms, rebuilds the skin barrier, and inhibits inflammation. These results suggest that the hydrogel integrating reactive oxygen species scavenging and FAK inhibition could serve as a promising skin dressing for synergistic AD treatment.

10. "liposome encapsulation has been used to promote the drug loading and sustained release of hydrophobic FAKi" Please provide references.
11. Please, mention that the "frequency sweep test" refers to shear testing using a rheometer.
-----------Farid Alisafaei, Ph.D. New Jersey Institute of Technology (NJIT) Reviewer #2 (Remarks to the Author): In this manuscript, Jia and colleagues developed an antibacterial self-healing hydrogel dressing that releases FAK inhibitors and ROS scavengers to treat atopic dermatitis. While ROS is known to be implicated in AD, this work shows for the first time that FAK signaling in the epidermis is activated by skin scratching, which led the authors to hypothesize that combining FAK inhibitors to dampen aberrant FAK signaling caused by scratching with ROS scavengers to reduce the inflammatory responses may be more effective strategy to treat AD compared to single factor treatments. Overall, the experiments are well designed and executed, and the in vivo data is compelling that this multifactorial approach is promising for treating this skin disorder. The manuscript is well written, but I have a few concerns and comments that need to be addressed to strengthen the findings and the impact of this study.

Major comments:
The methods on the animal experiments should be clarified. How was the MC-903 applied? Topically with a dressing, or by subcutaneous injection? After 14 days of injury stimulus, the skin lesions were covered with different hydrogels and 20µM MC-903 and 6 times of tape tears were applied daily. It's not clear if the hydrogels were removed from the lesion prior to the tape tears and MC-903 treatment, or if the gels remained in place. Also, was the same hydrogel used for the entire period of treatment, or was the hydrogel replaced with a fresh one daily?
The authors present in vitro data on the ROS scavenging properties of the hydrogels. But, given that the top keratinocyte layer is usually a dead layer of skin, how deep do the PDA nano particles penetrate the skin? Does scavenging ROS only manifest in the epidermis, or does it go beyond the basal epidermal layer?
It is not clear from the materials and methods how the release profile of FAKi was measured? Were the gels placed in an aqueous environment? Or were they placed in a transwell at a liquid interface, which would better approximate how the hydrogel releases its cargo when place topically on the skin?
The antibacterial properties of the hydrogel are convincingly demonstrated in vitro, but to what extent does the hydrogel eradicate bacterial growth in the skin, for instance after inoculation of the skin lesion with S. aureus? Is the treatment FAKi/ROS scavengers effective when a biofilm is present?
In Figure 3 (e and f): The HaCAT monolayer looks immature given that only half of the field of view is covered with cells. Cadherin stability and expression is strongly affected by cell density. How does stretch and stretch + HCF affect E-cadherin expression/localization in confluent monolayers? Images of static controls are needed to better gauge the effect of stretch and hydrogel treatment. How is Ecad deficiency measured?
Although the authors mention n values for each experiment, it is unclear whether 'n' stands for the number of technical replicates (i.e. duplicates or triplicates in one experiment) or biological replicates (i.e. different experiments). Given that standard deviation (SD) is reported rather than standard error of the mean (SEM), I presume that the in vitro experiments were performed once with n samples. The general consensus is to report data from at least three experimental repeats.
For in vivo experiments, each mouse can be considered as a separate experiment, which is why SEM rather than SD should be reported. Similarly, quantification of histological images should mention the number of sections/ROIs from how many mice that were used for the analysis.
The histology in figure 1, 6 and 7 is well executed, but the 'high magnification' images are too low in magnification to provide any additional informative compared to the low magnification images. The high magnification images should at least be two-fold higher or more in magnification as to better show where the signal for pFAK and fillagrin is at the cellular level. Also, arrows indicating positive signal will be helpful to the readers who aren't familiar with DAB stainings. Lastly, IGG controls for IF and IHC staining should be included, and the methods of quantification of the staining should be clarified.

Minor Comment
Can the authors elaborate on the self-healing properties of the hydrogel and include a few sentences on the mechanism of 'self-healing' works? Figure 7: A red line is used to demarcate the deficient are from the intact area, which is difficult to see for any reader whose color vision is impaired. A white line would be a better choice.
Line 76: What is mechanosensitization? I'm not sure if this word exists.
Typo's that need to be corrected: Line 17: synergistally Line 136 -"Lauri Acid" mistyped? Fig 6a -"Tap Scractch" mistyped? Throughout the paper, 'tap tears' should be 'tape tears' Reviewer #3 (Remarks to the Author): The manuscript by Jia et al, details the development of a hydrogel dressing for the potential use with atopic dermatitis.
Before the manuscript can be considered for publication there are a number of items that should be addressed. Line 45: 'Mechanical scratch is an important cause leading to …' how this sentence is worded does not make sense, please reword. Line 84: the statement regarding hydrogels and being used for their good biocompatibility. Using the term biocompatibility in general like this is incorrect. Biocompatible refers to the use of a certain material within a specific application. The general term of hydrogel does not specify the material used, not all hydrogels are biocompatible when used as in a skin dressing application. Line 110: refers to patients, this gives the impression that experiments were carried out in humans rather than mice. Please amend. Line 115: refers to rat scratching, though in vivo experiments were carried out in mice Line 119: refers to mast cell infiltration, through these sections were not stained to evaluate mast cell evaluation, please modify statement Line 174: states that the results show the hydrogels can be removed painlessly, though results shown do not demonstrate level of pain felt when hydrogels are removed from skin Line 272: refers to the expression of CCL-20 and TSLP (Fig 5d)

Response:
(1) We thank the reviewer for pointing out this. Many papers have highlighted FAK as a downstream convergence of mechanotransduction and many inflammatory signaling pathways. We have changed the vague description and added relevant references in the manuscript on Page 6.

On Page 6:
"FAK is at the point of biological convergence of mechanotransduction and many inflammatory signaling pathways [16][17][18] ." (2) We agree with the reviewers. As suggested by the reviewers, we used the selective non-muscle myosin II (NMII) inhibitor Blebbistatin to inhibit actomyosin contractility of HaCaT and repeated the stretching experiments. Many papers have reported that excessive inhibition of actomyosin contractility leads to reduced cell spreading, which would directly impair intercellular junctions. Thus, we first To verify whether FAK inhibition rescues E-cadherin deficiency by decreasing cellular contractility, we treated cells with the non-muscle myosin II inhibitor Blebbistatin.
Blebbistatin above 50 µM will lead to abnormal cell morphology and even reduced cell spreading, which will directly impair intercellular junctions. In contrast, 1,000 µg/ml of FAK-lipoLA (the maximum loading concentration of FAK-lipoLA in hydrogels) did not affect the morphology of HaCaT cells (Supplementary Fig. S6a,b). Therefore, 10 µM and 30 µM were chosen as working concentrations. Inhibition of actomyosin contractility by Blebbistatin rescues stretch-induced E-cadherin deficiency ( Supplementary Fig. S6c,d). However, compared to 30 µM, 10 µM Blebbistatin has a significantly reduced ability to rescue E-cadherin deficiency."  On Page 10: "To simulate the mechanical scratching in vitro, we used a customed stretching device to apply different cyclic stretching to HaCaT cells. Cells of the HC group (blank control, cultured with HC hydrogel extract) and the HCF group (cultured with HCF hydrogel extract) were separately seeded in the two compartments of a PDMS membrane to ensure that the two groups of cells are subjected to the same stretching (Fig. 5c)."

On Page 19:
"In vitro stretch experiments were carried out using a custom-made stretching device.
HaCaT cells were first seeded on a polydimethylsiloxane (PDMS) film for 24 h before stretching. Then, the culture medium was replaced with hydrogel extract (1 ml hydrogel soaking in 10 ml serum-free medium for 24 h). After culture for 12 h, cells were stretched at different strain levels. After stretching, the conditioned medium was obtained for ELISA tests, and cells were fixed using 4% paraformaldehyde for immunofluorescence staining." Figure 5C, did you also place a blank hydrogel (HC) on cells for the control case?

Comment #4. If you placed an HCF hydrogel on HaCaT cells for the stretching experiments in
Response: We are sorry for the confusion. We used HC hydrogel extract to culture HaCaT cells as the control group. To clarify this, we have revised related text. "It should be mentioned that although treatment with hydrogels relieved AD symptoms, in the HCPF group, skin lesions (Fig. 6), inflammatory markers (such as TSLP and IL-4, Fig. 7b,d) and intercellular junctions (Fig. 7g,i) are still not restored to the level of healthy mice. One possible reason is that the mice were exposed to a little dose of MC 903 and tape scratching during the hydrogel treatment period." c-g, CCL-20, TSLP, IgE, IL-4 and IL-13 levels in mouse tissues of each group, n= 5 mice. All data are shown as mean ± s.e.m.. *, **, ***and **** indicate P < 0.05, P < 0.01, P < 0.001 and P < 0.0001 compared using a two-tailed Student's t-test, respectively. Comment #8. It has been already shown that scavenging reactive oxygen species can alleviate AD symptoms. Therefore, I expected to see more comparisons between AD with and without mechanical deformations to show that FAK inhibition can further reduce the symptoms. However, in some cases, there is no comparison between AD with and without mechanical deformations. For example, why in Figure 6, there is no AD without scratch?
Response: We thank the reviewer for pointing out this. Comparison of H&E, pFAK and inflammatory factor between AD with and without mechanical scratch has been shown in Figure 1 and Supplementary Fig. S2. This narrative order is intended to make our story sound more logical, i.e., we first discovered the important role of scratching on AD inflammation and epidermal barriers and then carried out detailed studies afterwards. On the other hand, all treatments in Figure 6 were performed on AD mice with scratching. We think that it is not proper to compare these treatment results with scratching-free AD mice.
As suggested by the reviewers, we have added additional assessments of mast cell and epidermal barriers in Supplementary Fig. S2. There is no significant difference in mast cell infiltration in the ADscratch group compared to the unscratched group. However, by comparing the E-cadherin staining, we observed that the epidermis of unscratched AD skin is thickened with high fluorescence intensity of E-cadherin throughout the epidermis, while the fluorescence intensity of E-cadherin is significantly stratified in scratched AD skin. The filaggrin expression in the stratum corneum is also higher in the AD group than in the ADscratch group. These results indicate that scratching may cause the damage of epidermis barriers. To clarify this, we have added descriptions on Page 5.

On Page 5:
"Disruption of the epidermal barrier is one of the hallmarks of AD lesions and will increase skin sensitization to allergens. Thus, we evaluated two important skin barrier proteins, i.e., E-cadherin (mainly expressed in the epidermis) and filaggrin (mainly expressed in the stratum corneum). Immunofluorescent staining of E-cadherin shows obvious epidermal thickening in the AD and ADscratch group. Unscratched AD skin shows high fluorescence intensity of E-cadherin throughout the epidermis, while the fluorescence intensity of E-cadherin is significantly stratified in the ADscratch group ( Supplementary Fig. S2h,i). The filaggrin expression in the stratum corneum is also higher in the AD group than in the ADscratch group (Supplementary Fig. S2j-l). Besides, compared to healthy skin, pFAK is enriched in the AD group and significantly elevated in the epidermis of the ADscratch group (Fig. 2d). These results indicate that scratching may cause the damage of epidermis barriers, and pFAK is associated with AD and significantly enhanced by mechanical scratching." these regions are colored with nuclei. Therefore, we consider these regions as Ecadherin deficient regions. Furthermore, epidermal thickness as quantified by Ecadherin staining showed similar results to H&E staining (Supplementary Fig. S8h).

Supplementary
This suggests that the region where the nucleus is colored but E-cadherin fluorescence intensity is low is part of the epidermis. To better present the E-cadherin-deficient part, we used white lines to outline the cuticle and the demarcation between the deficient area and the intact area of E-cadherin (Fig. 7f).

Comment #10. "liposome encapsulation has been used to promote the drug loading and sustained release of hydrophobic FAKi" Please provide references.
Response: We thank the reviewer for pointing out this wrong description. There has been no report on liposome encapsulated FAK inhibitor. Our intent was that liposome encapsulation has been used to promote the drug loading and sustained release of hydrophobic drugs. We have changed the description and added references on Page 4.

On Page 4:
"while liposome encapsulation has been used to promote the drug loading and sustained release of hydrophobic drugs 33,34 " Comment #11. Please, mention that the "frequency sweep test" refers to shear testing using a rheometer.

Response:
We thank the reviewer for pointing out this. We have added this in the manuscript on Page 7.

On Page 7:
"Frequency sweep of shearing test by a rheometer revealed that the HC hydrogels have remarkable viscoelastic properties with a storage modulus of 1.24 ± 0.17 kPa, which is smaller than the modulus of human skin 41 ."

In this manuscript, Jia and colleagues developed an antibacterial self-healing hydrogel dressing that releases FAK inhibitors and ROS scavengers to treat atopic dermatitis.
While ROS is known to be implicated in AD, this work shows for the first time that FAK signaling in the epidermis is activated by skin scratching, which led the authors to hypothesize that combining FAK inhibitors to dampen aberrant FAK signaling caused by scratching with ROS scavengers to reduce the inflammatory responses may be more effective strategy to treat AD compared to single factor treatments. Overall, the experiments are well designed and executed, and the in vivo data is compelling that this multifactorial approach is promising for treating this skin disorder. The manuscript is well written, but I have a few concerns and comments that need to be addressed to strengthen the findings and the impact of this study.

Response:
We thank the reviewer for the encouraging comment. Response: We thank the reviewer for pointing out this. The MC 903 was directly applied dropwise to skin via pipette. Since the drug is dissolved by anhydrous ethanol, the drug solution will be rapidly absorbed after application. The hydrogels were removed from the lesion prior to the tape tears and MC 903 treatment and replaced with a fresh one daily. We have clarified these in the methods on Page 20.
On Page 20: "6-to 8-week-old male mice was shaved and treated with 45 µM MC 903 (dissolved in anhydrous ethanol) and mechanical scratching for 14 days (scheme in Fig. 6a). Briefly, after anesthetizing by 2% isoflurane, back skins of mice were tape-stripped with adhesive tapes (TegadermTM, 3M) for 10 times every day and then 40 µl 45 µM MC 903 were applied dropwise to skin via pipette. Since the drug is dissolved by anhydrous ethanol, the drug solution will be rapidly absorbed after application. During the modeling process, the injured area was constantly covered with gauze to avoid mechanical stimulation caused by the mice scratching. After 14 days, significant AD skin lesions were observed, and mice were randomly divided into 5 groups. "Besides, to test the in vivo ROS scavenging effect of hydrogels, fluorescent staining was performed on slides to detect 8-hydroxy-2'-deoxyguanosine (8-OHdG) ( Supplementary Fig. S8f), an oxidation product of deoxyguanosine residues in DNA 1,2 .
The results showed that HCP and HCPF hydrogels loaded with PDA NPs could effectively reduce DNA damage in the epidermis and dermis (Supplementary Fig.   S8g). The fluorescence intensity is slightly reduced in the HC and HCF groups relative to the ADscratch group, which may be due to the ROS scavenging effect of the dopamine groups in the HC hydrogels and the reduced acute inflammation by FAK inhibition."

Comment #3. It is not clear from the materials and methods how the release profile of
FAKi was measured? Were the gels placed in an aqueous environment? Or were they placed in a transwell at a liquid interface, which would better approximate how the hydrogel releases its cargo when place topically on the skin? Response: We thank the reviewer for pointing out this. We tested the release of FAK-lipoLA by placing the HCF hydrogel in a transwell at a liquid interface as suggested by the reviewer. To clarify this, we have revised relevant results in Figure 5b and added relevant descriptions in main text on Page 10 and Page 20.

On Page 10:
"We placed HCF hydrogels in a transwell to simulate the skin-hydrogel interface. The result showed that the FAKi-lipoLA in HCF could be released about 13% at 24 h and 18% at 72 h (Fig. 5b)."

On Page 19:
"Assessment of FAK-lipoLA release. Nile red-labeled FAK-lipoLA was used to prepare HCF hydrogels. HCF hydrogels were made into thick discs with 10 mm in diameter and 1 mm in thickness and placed in a transwell with polycarbonate membrane (pore size: 8 μm, Corning). Transwell was placed in a 12-well plate and DMEM was added to the plate well so that the liquid level of the medium in the transwell was approximately 0.5 mm. The medium was aspirated at different time points, and the fluorescence intensity of medium was tested using a microplate reader to calculate the release amount of FAK-lipoLA." (1) According to the reviewer's comments, we have stretched the HaCaT cells with a mature monolayer. We used HC hydrogel without FAKi-lipoLA as the blank control and set static (0% strain) groups. Interestingly, little E-cadherin staining was observed in the unstretched cells. Considering that in vivo skin tissues are continuously subjected to stretching at low strain and previous studies have reported that proper mechanical tension promotes intercellular E-cadherin formation, we stretched HaCaT cells at 5% strain for 4 h. The results showed that clear and intact intercellular E-cadherin is formed in the HC and HCF groups. Therefore, we chose cell monolayer with 5% stretch for 4 h to mimic normal skin. Based on this, we further stretched the cells at 30% strain for 4 h. After stretching, E-cadherin in HC group shows a significant deficiency, while this could be rescued in the HCF group.
These results demonstrate that FAKi-lipoLA can reduce E-cadherin deficiency in HaCaT cells under large mechanical stretching. We have revised the relevant figure and added related description on Page 10.
(2) E-cadherin deficiency is calculated as the ratio of the number of E-Cadherinuncolored intercellular junctions to the total number of intercellular junctions, i.e., if a cell has intercellular junctions with six surrounding cells and four of these intercellular junctions are not colored by E-cadherin, then the E-cadherin deficiency is 66.66%. We have added a description in Method section on Page 19.

On Page 10：
"E-cadherin is a major component of epidermal intercellular junctions, and its loss of expression correlates with impaired epidermal cell function and morphology 46 . Therefore, we examined the integrality of intercellular E-cadherin by immunofluorescence staining. Interestingly, there is little E-cadherin staining observed in the unstretched cells. Considering that in vivo skin tissues are continuously subjected to stretching at low strain and previous studies have reported that proper mechanical tension promotes intercellular E-cadherin formation 47,48 , we stretched HaCaT cells at 5% strain for 4 h. The results showed that clear and intact intercellular E-cadherin is formed in the HC and HCF groups. Therefore, we used cells with 5% stretching for 4 h to mimic normal skin. Based on this, we further stretched the cells at 30% strain for 4 h, which has been proven to cause mechanical damage to cells 49 . After stretching, Ecadherin in HC group shows a significant deficiency, while this could be rescued in the HCF group (Fig. 5d,e)."

On Page 19:
"E-cadherin deficiency is calculated as shown below:     "To evaluate the self-healing efficiency, we performed time-related strain sweep tests by applying a large shear strain (300%) mimicking mechanical scratching to destroy the HCPF hydrogels. The hydrogel was disrupted at 300% strain (the storage modulus G' decreases significantly to less than the loss modulus G"). When the strain is recovered to 1%, G' recovers rapidly within 20 s, indicating that the disrupted structure is restored (Fig. 3g). Adhesion experiments on the skin surface also showed that the dissected HCPF hydrogels could quickly heal within 10 s and withstand the large deformation at the joint (Fig. 3h)

Response:
We thank the reviewer for pointing out this.
(1) We have changed the description of "rat" to "mouse".
( (3) We have deleted the statement in Line 174 that hydrogel can be removed painlessly because no pain assessment was performed.
On Page 7: "Tests on human skin also showed that the HCPF hydrogels can adhere to tissue surfaces and withstand various skin deformation or joint movement and can be removed without residue." (4) We added the experimental data of CCL-20 in Figure 5d and have made corresponding changes in the main text on Page 10.
On Page 10: "For HC group, we observed that TNF-a, TSLP and CCL-20 secretions are significantly increased after 30% stretching, while treated with HCF extract significantly reduces their expressions under 30% stretching. Besides, for the 5% stretching groups, there is no difference between the HC and HCF groups, indicating that FAK-lipoLA does not affect the expression of inflammatory factors in the unstretched state (Fig. 5e)"

Figure 5 | Inhibition of FAK protects cells from mechanical damage in vitro.
f, TNF-a, TSLP and CCL-20 level in the culture medium of HaCaT cells after stretch.